Project Summary/Abstract Enzymes that utilize iron-containing cofactors for their activity catalyze a bewildering array of (often very difficult) chemical reactions that are fundamentally important to central life processes (e.g., DNA biosynthesis and repair, gene regulation, regulation of epigenetic inheritance, biosyntheses of a plethora of compounds with antibacterial and antifungal activities). Dysfunction of these enzymes is often associated with the onset of severe diseases, e.g. cancer, cardiovascular diseases, and diabetes. Strategies to harness the synthetic potential of these enzymes and to combat diseases associated with their dysfunction involves the rational manipulation of these processes on a molecular level. A prerequisite for this endeavor is a detailed knowledge of the underlying reaction mechanisms, in particular how the enzymes control the outcome of their reactions. The Bollinger/Krebs joint group specializes in combining transient-state rapid kinetic experiments with various spectroscopic (e.g. stopped-flow absorption, freeze-quench EPR and Mssbauer) and analytical (LC/MS) methods to monitor metalloenzyme reactions. In the last 15 years, their group has successfully studied many enzymes that require a mononuclear or a dinuclear non-heme-iron cofactor for activity by trapping and characterizing key reaction intermediates in their catalytic cycles. In particular, they identified high-spin Fe(IV)- oxo (ferryl) intermediates in several mononuclear non-heme-iron enzymes, mostly Fe(II)- and 2-oxo-glutarate- dependent (Fe/2OG) enzymes. The ferryl intermediate initiates substrate oxidation, typically by cleavage of an aliphatic C-H bond. The outcome of these reactions is diverse and includes hydroxylation (the default outcome), halogenation, desaturation, epimerization, and heterocyclization reactions. Many of these reactions are employed in the biosyntheses of medically important natural products. The current focus of research in the Bollinger/Krebs group aims at deciphering the factors that result in the diverse outcomes. The long-term goal of this research is to lay the foundation for the rational manipulation of these enzymes for biotechnological applications. The PI also has a long-standing collaboration with Squire Booker on mechanistic studies of Fe/S enzymes, in particular those that belong to the superfamily of radical S-adenosylmethionine (RS) enzymes. These enzymes use a [4Fe-4S] cluster to generate a canonical 5?-deoxy-adenos-5?-yl radical that initiates a wide variety of substrate oxidations, often by cleavage of an aliphatic C-H bond. The current focus of the collaborative research efforts on RS enzymes aim at delineating the reaction mechanisms of different reaction outcomes, viz sulfur insertion, methylation, methylthiolation, and desaturation.